Infection with Zika virus (ZIKV) in pregnancy has been associated with an increased incidence of a spectrum of birth defects collectively referred to as Congenital Zika Syndrome (CZS). We have demonstrated that the rhesus macaque is susceptible to ZIKV strains of French Polynesian, African, and American (Puerto Rican) origin, and our published and unpublished work has shown that vertical transmission in rhesus macaques is highly efficient: 5 of 5 fetuses, whether maternal infection was administered in the first or the third trimester, resulted in detectable vRNA in fetal tissues, and histopathology (chiefly inflammation) in fetal organs. Nonetheless, there is minimal understanding of the pathway by which ZIKV traverses the maternal-fetal barrier in vivo. Understanding how virus is afforded access to the fetal compartment will allow consideration of possible interventions. We have revised this proposal to study vertical transmission in the rhesus monkey during the first month after maternal infection to directly address this need, with the following Specific Aims. Specific Aim 1. To determine the pathway by which ZIKV transits the maternal-fetal interface in vertical transmission in vivo by assessing viral RNA burden in maternal, placental and fetal tissues. Specific Aim 2. To define the cellular impact of ZIKV infection by assessing tissue histopathology in parallel with virus localization at the maternal-fetal interface and in fetal tissues. Specific Aim 3. To define decidual leukocyte and placental Hofbauer cell populations with high-dimensional flow cytometry, and directly assess ZIKV infection with intracellular ZIKV antigen staining. With these Aims we will use the NHP model to comprehensively advance our understanding of the pathway and trajectory of vertical transmission of ZIKV. We will define the viral burden at the maternal-fetal interface during the processes leading to fetal infection. We will identify the cellular compartments which contain ZIKV protein and replicating virus. Finally, we will define the immunological responses in the maternal decidua and the fetal placenta during vertical transmission. To accomplish these goals, we will work with a team of expert NHP virologists who have established the macaque model of ZIKV infection, and pathologists and reproductive immunologists who can provide expert and comprehensive assessment of both maternal and fetal outcomes of ZIKV infection. The development of therapies requires insight into pathogenesis. By defining the pathway(s) of vertical transmission, we will have established a relevant NHP experimental platform for testing approaches to interrupt vertical transmission and the development of CZS in human infants.